Classical alkylating agents evidently produce their therapeutic as well as their mutagenic and carcinogenic effects by attacking the DNA of target cells. The newer nitrosourea compounds, bis-chloroethyl nitrosoureas (BCNU) and chloroethyl cyclohexyl nitrosourea (CCNU), alkylate nucleic acids to a lesser extent, but the specificity of the reaction appears to be different. We will study the chemistry of these reactions, determining the sites of base substitution and the effects of reaction on the physical structure of synthetic polynucleotides. These polymers have a relatively simple structure so that the chemical effects of individual base alkylations can be determined precisely. Furthermore, they can be used as templates for replication by RNA polymerase. Accordingly, it is possible to compare the behavior of alkylated and unalkylated templates to determine which reactions of BCNU and CCNU are biologically significant. By comparing these results with our earlier data on classical alkylating agents, we will look for explanations of the clinically-observed differences in these compounds. Although major emphasis will be placed on the therapeutic value of these agents, some attention will be paid to possible mutagenic and carcinogenic side reactions.